The overall goal of this project is to discover novel in vivo chemical probes that are capable of potently and selectively disrupting the CXCL12/CXCR4 protein-protein interface. The chemokine CXCL12 and its G protein-coupled receptor CXCR4 are high-priority clinical targets because of their involvement in metastatic cancers (also implicated in autoimmune disease and cardiovascular disease). Because chemokines simultaneously interact with two distinct sites to bind and activate their receptors, both the GPCR and its ligand are potential targets for inhibition by small molecules. Recently, we developed a novel structure-based hybrid in silico/NMR screening strategy and identified a CXCL12 ligand that occludes the receptor recognition site. We optimized the initial hit by synthesizing a panel of tetrazole derivatives using a fragment-based approach. The best molecules in this series demonstrated a Kd of 13 mM (LE of 0.33) by 2D NMR spectroscopy and completely inhibited CXCL12-mediated chemotaxis of CXCR4-expressing cells. Significantly, we solved the first X-ray co-crystal structure of chemokine CXCL12 with one of our tetrazole fragments (a Kd of 41 mM by 2D NMR spectroscopy, LE of 0.28) at 1.8 A resolution. In a parallel screening effort by our collaborators at UCSF, a new CXCR4 antagonist was identified by docking over three million molecules to the crystal structure of CXCR4 and confirmed with an IC50 of 57 mM by calcium flux assay. The hit exhibited an IC50 value of 310 nM by radioligand displacement of 50 pM [I125]-CXCL12, LE of 0.36, and efficacy in blocking cellular chemotaxis. We propose to develop these validated CXCL12 and CXCR4 inhibitors into in vivo chemical probes for use in diagnosis and treatment of metastatic disease. The aims of this grant application are: 1) design, synthesize and optimize CXCL12 chemokine inhibitors. 2) Design, synthesize and optimize CXCR4 receptor antagonists. 3) Develop potent and selective inhibitors as in vivo chemical probes using robust bioassays and structural determination of co-complexes using both X-ray crystallography and 2D NMR spectroscopy. Because chemokine signaling plays a key role in cancer cell migration, both CXCL12 inhibitors and CXCR4 antagonists are likely to find utility as novel therapeutic agents for metastatic disease. Finally, we will assess he in vivo efficacy of optimized compounds using animal models for CXCR4-directed metastatic cancer as described in our recent publication in PNAS. This is a key prerequisite for PAR-12-060.